Spinocerebellar ataxia type-6 (SCA6) is a dominantly inherited ataxia caused by a polyglutamine (polyQ)- encoding CAG repeat expansion near the 3'-end of the Cav2.1 calcium channel gene, CACNA1a. Selective loss of cerebellar Purkinje neurons is the pathological hallmark of SCA6, a slowly progressive and debilitating disease for which there is no treatment. The preclinical studies proposed here will test a novel splice-isoform- specific RNA interference (SIS-RNAi) therapy approach for SCA6, using a genetically accurate mouse model of the disease. Alternative splicing at the 3'-end of the Cav2.1 transcript produces mRNA isoforms that encode two classes of Cav2.1 variants, one lacking and one containing the polyQ domain near the c-terminal end of the protein. In SCA6, recent studies have shown an aberrant increase in the levels of splice isoforms encoding for mutant (expanded) polyQ-containing Cav2.1 variants. Levels of toxic polyQ-containing Cav2.1 variants appear to increase only in cerebellar Purkinje neurons, potentially explaining their selective degeneration in SCA6. The overall hypothesis is that a novel SIS-RNAi approach developed by our research group will selectively suppress expression of polyQ-containing Cav2.1 variants in Purkinje neurons and thus will prove to be of significant therapeutic benefit in SCA6. Aim 1 will establish the capacity of recombinant adeno-associated (rAAV)-delivered SIS-RNAi molecules to selectively suppress the expression of human, polyQ-encoding Cav2.1 splice-isoforms in Purkinje neurons of an SCA6 knock-in mouse model. SCA6 knock-in mice express expanded polyQ-encoding Cav2.1 transcripts under the control of the endogenous mouse Cav2.1 promoter. Importantly, these mice faithfully recapitulate motor and histo-pathological features observed in human SCA6. Aim 2 will examine the molecular and behavioral effects of long-term rAAV-SIS-RNAi expression on the progressive neurological dysfunction observed in SCA6 knockin mice. The primary impact of the proposed studies is that they are expected to provide preclinical data supporting the development of SIS-RNAi as therapy for humans with SCA6. Moreover, they will highlight the potential of SIS-RNAi as a therapeutic strategy in other hereditary diseases and forge novel reagents useful to our understanding of Cav2.1 channel function and regulation.